Previous work under this project has established that multiple forms of proteoglycans exist in the corneal stroma of adult rabbits (two proteokeratan sulfates and two proteodermatan sulfates). The chemical characterization of these was extended to studies on their biosynthesis in vivo and in organ cultures of intact corneas by incorporation of radiolabeled precursors. Similar chemical work on bovine corneas (done to permit correlation with results from other laboratories) indicates that essentially the same multiple forms are present, and this is probably a general Phenomenon. Human corneas are being collected for a parallel study. Monoclonal antibodies have now been prepared against some of the proteoglycans (collaboration with Dr. Nirmala SundarRaj, Dept. of Ophthalmology, Univ. of Pittsburgh Sch. of Medicine). Development of a tissue is often accompanied by progressive changes in the properties of any given class of macromolecules (e.g., collagen, proteoglycans). The various stages have been called "isoforms," and such series of changes may be important in the mechanism of development and may extend through maturity to contribute to the aging process. There is already evidence that the proteoglycan composition of cornea changes markedly during fetal and postnatal development. The following areas of interest will now be examined. 1) The physiological function of proteoglycans in corneal stroma will be investigated by measurement of their tendency to interact with and bind to other components of the extracellular matrix, such as collagen, fibronectin, and laminin. Existing methods of affinity chromatography and immunochemical measurements will be used. 2) The onset of transparency during corneal development will be correlated with measurement of the sizes and compositions of the proteoglycans by means of labeling with precursors of carbohydrate and protein (collaboration with Dr. Charles Cintron, Eye Research Institute, Boston, MA), and immunochemical properties will also be compared. Preliminary experiments have already shown differences in the sizes and degree of sulfation of the various components synthesized by fetal and postnatal corneas before they become transparent. Since there are certain parallels between developmental changes and changes in proteoglycans that occur during healing and restoration of transparency in injured corneas, analysis of the behavior of these corneal macromolecules may also help understanding of the healing process.